title: Hypoxic conditions during expansion culture prime human mesenchymal stromal precursor cells for chondrogenic differentiation in three-dimensional cultures.
abstract: Cell-based approaches using mesenchymal stromal precursor cells (MSCs) for the regeneration of intervertebral discs are attracting increased interest, even though the intervertebral disc is a very demanding environment. Implanted cells eventually face acidic pH, hypoxia, and a lack  of nutrients. While the regenerative potential of MSCs for skeletal tissues has been well described, it is still questionable whether human MSCs can be prepared for prolonged survival and proper functioning and whether they can differentiate under the adverse conditions encountered in the  disc. Here we examined the influence of hypoxia during expansion and differentiation on the chondrogenesis of MSCs. Chondrogenic differentiation was performed in in situ solidifying gelatin hydrogels, which represent a suitable matrix for delivering and anchoring cells within the disc tissue.  To consider limitations in nutrition in the intervertebral disc, differentiation was performed at low cell concentrations in the gelatin hydrogels. Standard high-density micromass cultures served as reference controls. To determine the quality of chondrogenesis we analyzed typical marker molecules  such as collagen types I, II, X, Sox-9, MIA, and aggrecan mRNA using RT-qPCR and determined protein deposition by histological stainings and biochemical methods. We could demonstrate that in gelatin-based hydrogels chondrogenic differentiation of human MSCs is possible at low cell concentrations.  The quality of chondrogenic differentiation could be improved by hypoxia. Best results were obtained when the entire in vitro process, including MSC expansion and subsequent differentiation, was done under hypoxic conditions. MSCs that were expanded under reduced oxygen tension were primed  for a chondrogenic differentiation.
authors.affiliation: NMI Natural and Medical Sciences Institute, University of Tübingen, Reutlingen, Germany.
authors.forename: Jana
authors.initials: J
authors.lastname: Müller
authors.forename: Karin
authors.initials: K
authors.lastname: Benz
authors.forename: Michael
authors.initials: M
authors.lastname: Ahlers
authors.forename: Christoph
authors.initials: C
authors.lastname: Gaissmaier
authors.forename: Jürgen
authors.initials: J
authors.lastname: Mollenhauer
mesh_terms: D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D015687:Cell Hypoxia / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D020219:Chondrogenesis / Q000235:genetics / Q000502:physiology*; D024042:Collagen Type I / Q000378:metabolism; D024043:Collagen Type II / Q000378:metabolism; D024121:Collagen Type X / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D009363:Neoplasm Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055758:SOX9 Transcription Factor / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology*
chemical_list: D024042:Collagen Type I; D024043:Collagen Type II; D024121:Collagen Type X; D016326:Extracellular Matrix Proteins; C090059:MIA protein, human; D009363:Neoplasm Proteins; D055758:SOX9 Transcription Factor